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Importance of Shank3 Protein in Regulating Metabotropic Glutamate Receptor 5 (mGluR5) Expression and Signaling at Synapses

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Verpelli, Chiara, Dvoretskova, Elena, Vicidomini, Cinzia, Rossi, Francesca, Chiappalone, Michela, Schoen, Michael, Di Stefano, Bruno, Mantegazza, Renato, Broccoli, Vania, Böckers, Tobias M., Dityatev, Alexander, Sala, Carlo·2011·Journal of Biological Chemistry
DOI 10.1074/jbc.m111.258384PMC PMC3186429
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Abstract

Shank3/PROSAP2 gene mutations are associated with cognitive impairment ranging from mental retardation to autism. Shank3 is a large scaffold postsynaptic density protein implicated in dendritic spines and synapse formation; however, its specific functions have not been clearly demonstrated. We have used RNAi to knockdown Shank3 expression in neuronal cultures and showed that this treatment specifically reduced the synaptic expression of the metabotropic glutamate receptor 5 (mGluR5), but did not affect the expression of other major synaptic proteins. The functional consequence of Shank3 RNAi knockdown was impaired signaling via mGluR5, as shown by reduction in ERK1/2 and CREB phosphorylation induced by stimulation with (S)-3,5-dihydroxyphenylglycine (DHPG) as the agonist of mGluR5 receptors, impaired mGluR5-dependent synaptic plasticity (DHPG-induced long-term depression), and impaired mGluR5-dependent modulation of neural network activity. We also found morphological abnormalities in the structure of synapses (spine number, width, and length) and impaired glutamatergic synaptic transmission, as shown by reduction in the frequency of miniature excitatory postsynaptic currents (mEPSC). Notably, pharmacological augmentation of mGluR5 activity using 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)-benzamide as the positive allosteric modulator of these receptors restored mGluR5-dependent signaling (DHPG-induced phosphorylation of ERK1/2) and normalized the frequency of mEPSCs in Shank3-knocked down neurons. These data demonstrate that a deficit in mGluR5-mediated intracellular signaling in Shank3 knockdown neurons can be compensated by 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)-benzamide; this raises the possibility that pharmacological augmentation of mGluR5 activity represents a possible new therapeutic approach for patients with Shank3 mutations.

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FIGURE 1. Knocking down Shank3 reduces mGluR5 expression. A, histogram showing mean ± S.E. for four independent experiments, of mRNA levels of the indicated proteins (normalized against those of uninfected neurons) in hippocampal neurons infected or not with a lentivirus expressing shShank3 or control shRNA (shCtrl); the level of Shank3 mRNA was significantly lower in the shShank3-infected neurons than in uninfected or shCtrl-infected neurons, *, p < 0.01, Student's t test. B, Western blot of hippocampal neurons infected with 3 and 6 μl of lentivirus preparations expressing shShank3 or shCtrl and Shank3; Shank3 was detected using rabbit H-160 anti-Shank3 antibody. The four major bands recognized by the antibody are indicated by the arrows. C, Western blot of hippocampal neurons infected at 7 DIV with a lentivirus expressing shShank3 or shCtrl, as indicated above the panels, and analyzed with the antibodies against the proteins indicated on the left side of the panels. D, histogram showing mean ± S.E. for four independent experiments, of protein levels (normalized against those of the uninfected neurons) in the hippocampal neurons infected with a lentivirus expressing shShank3 or shCtrl. The expression levels of Shank3 and mGluR5 were significantly lower in the shShank3-infected neurons than in uninfected and shCtrl-infected neurons; *, p < 0.01, Student's t test. E, Western blot of synaptosomes obtained from hippocampal neurons infected with a lentivirus expressing shShank3 or shCtrl and analyzed with the antibodies against the proteins indicated on the left side of the panel. F, histogram showing mean ± S.E. for three independent experiments, of protein levels (normalized against those of the shCtrl infected neurons) in synaptosomes obtained from hippocampal neurons infected with a lentivirus expressing shShank3 or shCtrl. The expression levels of Shank3 and mGluR5 were significantly lower in the shShank3-infected neurons than in uninfected and shCtrl-infected neurons; *, p < 0.01, Student's t test.
FIGURE 1. Knocking down Shank3 reduces mGluR5 expression. A, histogram showing mean ± S.E. for four independent experiments, of mRNA levels of the indicated proteins (normalized against those of uninfected neurons) in hippocampal neurons infected or not with a lentivirus expressing shShank3 or control shRNA (shCtrl); the level of Shank3 mRNA was significantly lower in the shShank3-infected neurons than in uninfected or shCtrl-infected neurons, *, p < 0.01, Student's t test. B, Western blot of hippocampal neurons infected with 3 and 6 μl of lentivirus preparations expressing shShank3 or shCtrl and Shank3; Shank3 was detected using rabbit H-160 anti-Shank3 antibody. The four major bands recognized by the antibody are indicated by the arrows. C, Western blot of hippocampal neurons infected at 7 DIV with a lentivirus expressing shShank3 or shCtrl, as indicated above the panels, and analyzed with the antibodies against the proteins indicated on the left side of the panels. D, histogram showing mean ± S.E. for four independent experiments, of protein levels (normalized against those of the uninfected neurons) in the hippocampal neurons infected with a lentivirus expressing shShank3 or shCtrl. The expression levels of Shank3 and mGluR5 were significantly lower in the shShank3-infected neurons than in uninfected and shCtrl-infected neurons; *, p < 0.01, Student's t test. E, Western blot of synaptosomes obtained from hippocampal neurons infected with a lentivirus expressing shShank3 or shCtrl and analyzed with the antibodies against the proteins indicated on the left side of the panel. F, histogram showing mean ± S.E. for three independent experiments, of protein levels (normalized against those of the shCtrl infected neurons) in synaptosomes obtained from hippocampal neurons infected with a lentivirus expressing shShank3 or shCtrl. The expression levels of Shank3 and mGluR5 were significantly lower in the shShank3-infected neurons than in uninfected and shCtrl-infected neurons; *, p < 0.01, Student's t test.CC BY-NC · PubMed Central
FIGURE 2. A, confocal microscopy images of hippocampal neurons infected or not at 7 DIV with a lentivirus expressing shShank3 or shCtrl and stained after 1 week with antibodies specific for Shank3, synaptophysin, and PSD-95 (as indicated above the panels). Scale bar = 5 μm. B, histogram showing mean ± S.E. for four independent experiments, of numbers of Shank3, synaptophysin, and PSD-95 clusters in hippocampal neurons infected with lentivirus expressing shShank3 or shCtrl; at least five neurons were considered for each experiment. The number of Shank3 clusters was significantly lower in the shShank3-infected neurons than in uninfected and shCtrl-infected neurons; *, p < 0.01, Student's t test. C, confocal microscopy images showing dendritic spine morphology in hippocampal neurons infected with shShank3. Hippocampal neurons at 7 DIV were transfected with GFP-containing shCtrl or shShank3 cDNAs, plus DsRed-expressing cDNA. After a week, neurons were fixed and stained for GFP and DsRed. D, quantification of number (per 10 μm), length, and width of dendritic spines (± S.E.). Over 14 transfected neurons from four independent experiments were measured for each transfection. Dendritic spine number, length, and width were significantly different in the shShank3-transfected and shCtrl-transfected neurons; **, p < 0.05; *, p < 0.01, Student's t test. Scale bar = 10 μm.
FIGURE 2. A, confocal microscopy images of hippocampal neurons infected or not at 7 DIV with a lentivirus expressing shShank3 or shCtrl and stained after 1 week with antibodies specific for Shank3, synaptophysin, and PSD-95 (as indicated above the panels). Scale bar = 5 μm. B, histogram showing mean ± S.E. for four independent experiments, of numbers of Shank3, synaptophysin, and PSD-95 clusters in hippocampal neurons infected with lentivirus expressing shShank3 or shCtrl; at least five neurons were considered for each experiment. The number of Shank3 clusters was significantly lower in the shShank3-infected neurons than in uninfected and shCtrl-infected neurons; *, p < 0.01, Student's t test. C, confocal microscopy images showing dendritic spine morphology in hippocampal neurons infected with shShank3. Hippocampal neurons at 7 DIV were transfected with GFP-containing shCtrl or shShank3 cDNAs, plus DsRed-expressing cDNA. After a week, neurons were fixed and stained for GFP and DsRed. D, quantification of number (per 10 μm), length, and width of dendritic spines (± S.E.). Over 14 transfected neurons from four independent experiments were measured for each transfection. Dendritic spine number, length, and width were significantly different in the shShank3-transfected and shCtrl-transfected neurons; **, p < 0.05; *, p < 0.01, Student's t test. Scale bar = 10 μm.CC BY-NC · PubMed Central
FIGURE 3. Knockdown of Shank3 alters the mGluR5 pathway. A, Western blot of hippocampal neurons infected at 7 DIV with shCtrl or shShank3. After 1 week, neurons were treated with 100 μm DHPG, 100 μm NMDA, or 50 mm KCl for 30 min, as indicated above the panels, and analyzed Shank3, pERK1/2, ERK1/2, pCREB, and CREB expression, as indicated on the left of the panels. B, histogram showing mean ± S.E., for five independent experiments, of pERK1/2 and pCREB levels (normalized against total ERK and CREB and untreated control (NT) values). The levels of pERK1/2 and pCREB were significantly lower in the shShank3-infected neurons than in the shCtrl-infected neurons after DHPG treatment; *, p < 0.01, Student's t test. C, confocal microscopy images of hippocampal neurons transfected at 7 DIV with shCtrl, shShank3, or shShank3 plus Shank3r, as indicated on the left side of the panels, and treated on 14 DIV with 100 μm DHPG or 50 mm KCl, as indicated above the panels (NT, untreated). The neurons were fixed and stained for GFP and pERK1/2. D, histogram showing mean ± S.E. values for pERK1/2 signals in each condition of transfection and treatment quantified as described under “Experimental Procedures.” The level of pERK1/2 after DHPG treatment was significantly lower in the shShank3-transfected neurons than in the shCtrl- and shShank3 plus Shank3r-transfected neurons; *, p < 0.05, Student's t test. Scale bar = 20 μm.
FIGURE 3. Knockdown of Shank3 alters the mGluR5 pathway. A, Western blot of hippocampal neurons infected at 7 DIV with shCtrl or shShank3. After 1 week, neurons were treated with 100 μm DHPG, 100 μm NMDA, or 50 mm KCl for 30 min, as indicated above the panels, and analyzed Shank3, pERK1/2, ERK1/2, pCREB, and CREB expression, as indicated on the left of the panels. B, histogram showing mean ± S.E., for five independent experiments, of pERK1/2 and pCREB levels (normalized against total ERK and CREB and untreated control (NT) values). The levels of pERK1/2 and pCREB were significantly lower in the shShank3-infected neurons than in the shCtrl-infected neurons after DHPG treatment; *, p < 0.01, Student's t test. C, confocal microscopy images of hippocampal neurons transfected at 7 DIV with shCtrl, shShank3, or shShank3 plus Shank3r, as indicated on the left side of the panels, and treated on 14 DIV with 100 μm DHPG or 50 mm KCl, as indicated above the panels (NT, untreated). The neurons were fixed and stained for GFP and pERK1/2. D, histogram showing mean ± S.E. values for pERK1/2 signals in each condition of transfection and treatment quantified as described under “Experimental Procedures.” The level of pERK1/2 after DHPG treatment was significantly lower in the shShank3-transfected neurons than in the shCtrl- and shShank3 plus Shank3r-transfected neurons; *, p < 0.05, Student's t test. Scale bar = 20 μm.CC BY-NC · PubMed Central
FIGURE 4. Shank3 mutations found in autistic patients do not rescue shShank3-induced defects in the mGluR5 pathway. A, Western blot of COS-7 cells transfected with HA-Shank3, GFP-Shank3r, GFP-Shank3R87Cr, or GFP-Shank3InsGr plus or minus shShank3, as indicated above the panels, and analyzed with anti-HA, anti-GFP, or anti-tubulin antibodies. B, confocal microscopy images of hippocampal neurons transfected at 7 DIV with shShank3 + Shank3R87Cr or shShank3 + Shank3InsGr, as indicated on the left side of the panels, and treated or not at 14 DIV with 100 μm DHPG or 50 mm KCl, as indicated above the panels; the cells were fixed and stained for GFP and pERK1/2. C, histogram showing mean ± S.E. values for pERK1/2 in each condition of transfection and treatment quantified as described under “Experimental Procedures.” The level of pERK1/2 was significantly lower after DHPG treatment in the shShank3-, shShank3 + Shank3R87Cr-, and shShank3 + Shank3InsGr-transfected neurons than in the shCtrl-transfected neurons; *, p < 0.05, Student's t test. Scale bar = 20 μm.
FIGURE 4. Shank3 mutations found in autistic patients do not rescue shShank3-induced defects in the mGluR5 pathway. A, Western blot of COS-7 cells transfected with HA-Shank3, GFP-Shank3r, GFP-Shank3R87Cr, or GFP-Shank3InsGr plus or minus shShank3, as indicated above the panels, and analyzed with anti-HA, anti-GFP, or anti-tubulin antibodies. B, confocal microscopy images of hippocampal neurons transfected at 7 DIV with shShank3 + Shank3R87Cr or shShank3 + Shank3InsGr, as indicated on the left side of the panels, and treated or not at 14 DIV with 100 μm DHPG or 50 mm KCl, as indicated above the panels; the cells were fixed and stained for GFP and pERK1/2. C, histogram showing mean ± S.E. values for pERK1/2 in each condition of transfection and treatment quantified as described under “Experimental Procedures.” The level of pERK1/2 was significantly lower after DHPG treatment in the shShank3-, shShank3 + Shank3R87Cr-, and shShank3 + Shank3InsGr-transfected neurons than in the shCtrl-transfected neurons; *, p < 0.05, Student's t test. Scale bar = 20 μm.CC BY-NC · PubMed Central
FIGURE 5. Knockdown of Shank3 expression reduces the frequency of mEPSCs in cultured hippocampal neurons, whereas reintroduction of shShank3-resistant Shank3 restores the frequency of mEPSCs. A, representative mEPSCs recorded on 14 DIV from control neurons (shCtrl) and after knockdown of Shank3 (shShank3), at a holding potential of −60 mV in the presence of 0.5 μm TTX and 50 μm picrotoxin. B, averaged mEPSCs from a control neuron (left) and from a shShank3-treated neuron (right). C-F, summary histograms of mEPSC parameters in control (n = 16) and shShank3-treated (n = 19) neurons. Data are from seven culture preparations. There is no significant difference in the mEPSC amplitude, rise, or decay time. The frequency of mEPSCs in shShank3-treated neurons is significantly decreased as compared with control neurons; **, p < 0.005; Student's t test). G, representative mEPSCs recorded on 14 DIV from control neurons (shCtrl + mock), after knockdown of Shank3 (shShank3 + mock) at 8 DIV, and after transfection of Shank3 knocked-down neurons with shShank3-resistant Shank3 at 11 DIV (shShank3 + Shank3r). H and I, summary histograms of the amplitude (H) and frequency (I) of mEPSCs in shCtrl + mock (n = 12 cells), shShank3 + mock (n = 11 cells), and shShank3 + Shank3r (n = 14 cells) treated groups. Data are from four independent experiments. ANOVA revealed a difference in frequency between the three groups (p < 0.01). The frequency of mEPSCs was decreased in shShank3 + mock-treated neurons (*, p < 0.05, Dunn's test) and recovered in shShank3 + Shank3r-treated cells (*, p < 0.05, Dunn's test). Data are presented as mean ± S.E.
FIGURE 5. Knockdown of Shank3 expression reduces the frequency of mEPSCs in cultured hippocampal neurons, whereas reintroduction of shShank3-resistant Shank3 restores the frequency of mEPSCs. A, representative mEPSCs recorded on 14 DIV from control neurons (shCtrl) and after knockdown of Shank3 (shShank3), at a holding potential of −60 mV in the presence of 0.5 μm TTX and 50 μm picrotoxin. B, averaged mEPSCs from a control neuron (left) and from a shShank3-treated neuron (right). C-F, summary histograms of mEPSC parameters in control (n = 16) and shShank3-treated (n = 19) neurons. Data are from seven culture preparations. There is no significant difference in the mEPSC amplitude, rise, or decay time. The frequency of mEPSCs in shShank3-treated neurons is significantly decreased as compared with control neurons; **, p < 0.005; Student's t test). G, representative mEPSCs recorded on 14 DIV from control neurons (shCtrl + mock), after knockdown of Shank3 (shShank3 + mock) at 8 DIV, and after transfection of Shank3 knocked-down neurons with shShank3-resistant Shank3 at 11 DIV (shShank3 + Shank3r). H and I, summary histograms of the amplitude (H) and frequency (I) of mEPSCs in shCtrl + mock (n = 12 cells), shShank3 + mock (n = 11 cells), and shShank3 + Shank3r (n = 14 cells) treated groups. Data are from four independent experiments. ANOVA revealed a difference in frequency between the three groups (p < 0.01). The frequency of mEPSCs was decreased in shShank3 + mock-treated neurons (*, p < 0.05, Dunn's test) and recovered in shShank3 + Shank3r-treated cells (*, p < 0.05, Dunn's test). Data are presented as mean ± S.E.CC BY-NC · PubMed Central
FIGURE 6. Impairment of DHPG-induced LTD and reduction in cell surface expression of the AMPAR subunit GluR1 in shShank3-treated hippocampal neurons. A, examples of mEPSCs recorded immediately before (left) and 30 min after (right) DHPG application. Miniature EPSCs were recorded at a holding potential of −60 mV in the presence of 0.5 μm TTX and 50 μm picrotoxin. To isolate mGluR-mediated long term depression, 50 μm APV was co-applied with 100 μm DHPG or applied alone as a control. A, top, in shCtrl-treated cultures, 100 μm DHPG elicited a persistent decrease in mEPSC frequency. A, bottom, in shShank3-treated neurons, 100 μm DHPG did not induce a decrease in mEPSC frequency. B, histogram showing mean ± S.E. % mEPSC amplitude in shCtrl or in shShank3-treated neurons stimulated with DHPG. C, effect of DHPG on mEPSC frequency in shCtrl (black circles, n = 7 neurons) and in shShank3-treated neurons (gray circles, n = 7 neurons). Data presented are mean ± S.E. from three culture preparations. The frequency of mEPSCs during the first 5 min of recordings (i.e. before application of DHPG) was set to 100%. D, summary plot showing changes in mean mEPSC frequency 30–35 min after application of DHPG; *, p < 0.05, Student's t test. E, examples of confocal images of cell surface immunostaining of GluR1 in hippocampal neurons at 14 DIV. Neurons were treated with 100 μm DHPG (+DHPG) for 10 min, or not treated (−DHPG). E, top). There is a reduction in cell surface expression of GluR1 in control neurons (shCtrl) after DHPG treatment. E, bottom, in shShank3-treated neurons, the number of GluR1-immunoreactive clusters on the surface of dendrites was reduced, and treatment with DHPG did not affect the surface expression of GluR1. F, summary plot of GluR1 cell surface distribution in shCtrl and shShank3-treated neurons after DHPG treatment. The diagram summarizes data from five culture preparations; #, p < 0.05, Student's t test; *, p < 0.05, paired t test. Data are presented as mean ± S.E.
FIGURE 6. Impairment of DHPG-induced LTD and reduction in cell surface expression of the AMPAR subunit GluR1 in shShank3-treated hippocampal neurons. A, examples of mEPSCs recorded immediately before (left) and 30 min after (right) DHPG application. Miniature EPSCs were recorded at a holding potential of −60 mV in the presence of 0.5 μm TTX and 50 μm picrotoxin. To isolate mGluR-mediated long term depression, 50 μm APV was co-applied with 100 μm DHPG or applied alone as a control. A, top, in shCtrl-treated cultures, 100 μm DHPG elicited a persistent decrease in mEPSC frequency. A, bottom, in shShank3-treated neurons, 100 μm DHPG did not induce a decrease in mEPSC frequency. B, histogram showing mean ± S.E. % mEPSC amplitude in shCtrl or in shShank3-treated neurons stimulated with DHPG. C, effect of DHPG on mEPSC frequency in shCtrl (black circles, n = 7 neurons) and in shShank3-treated neurons (gray circles, n = 7 neurons). Data presented are mean ± S.E. from three culture preparations. The frequency of mEPSCs during the first 5 min of recordings (i.e. before application of DHPG) was set to 100%. D, summary plot showing changes in mean mEPSC frequency 30–35 min after application of DHPG; *, p < 0.05, Student's t test. E, examples of confocal images of cell surface immunostaining of GluR1 in hippocampal neurons at 14 DIV. Neurons were treated with 100 μm DHPG (+DHPG) for 10 min, or not treated (−DHPG). E, top). There is a reduction in cell surface expression of GluR1 in control neurons (shCtrl) after DHPG treatment. E, bottom, in shShank3-treated neurons, the number of GluR1-immunoreactive clusters on the surface of dendrites was reduced, and treatment with DHPG did not affect the surface expression of GluR1. F, summary plot of GluR1 cell surface distribution in shCtrl and shShank3-treated neurons after DHPG treatment. The diagram summarizes data from five culture preparations; #, p < 0.05, Student's t test; *, p < 0.05, paired t test. Data are presented as mean ± S.E.CC BY-NC · PubMed Central
FIGURE 7. Knockdown of Shank3 impairs DHPG-induced modulation of neuronal network activity. A, representative 60-electrode recordings of activity of cultured mouse neurons before and 5 min after application of 100 μm DHPG. Each horizontal line corresponds to one electrode; short vertical intervals correspond to detected action potentials. The duration of each recording is 30 s. Following application of 100 μm DHPG, a greater increase in the number of bursts occurs in the shCtrl-treated cultures compared with the shShank3-treated cultures. B-E, changes in the mean firing rate (B), bursting rate (C), intra-burst firing rate (D), and percentage of out-burst spikes (E) for active electrodes after the application of DHPG to shCtrl- and shShank3-treated cultures. There were no differences between shCtrl- and shShank3-treated groups in baseline activity; therefore, the values of parameters estimated under the baseline condition were set to 100% for each culture. Two-way ANOVA with repeated measures (DHPG concentrations of 1, 10, and 100 μm) revealed a significant effect of DHPG (p < 0.01), but not of shShank3 or shShank3 + DHPG on the mean firing rate (B). Significant effects of DHPG (p < 0.01) and shShank3 (p = 0.05) on the mean bursting rate were detected in C, and significant effects of shShank3 + DHPG (p < 0.05) on the intra-burst firing rate (D) and % of out-burst spikes (E) were detected. *, p < 0.05; **, p < 0.01, Holm-Sidak post hoc t test, significant differences between shCtrl- (n = 10 cultures) and shShank3-treated cultures (n = 7 cultures).
FIGURE 7. Knockdown of Shank3 impairs DHPG-induced modulation of neuronal network activity. A, representative 60-electrode recordings of activity of cultured mouse neurons before and 5 min after application of 100 μm DHPG. Each horizontal line corresponds to one electrode; short vertical intervals correspond to detected action potentials. The duration of each recording is 30 s. Following application of 100 μm DHPG, a greater increase in the number of bursts occurs in the shCtrl-treated cultures compared with the shShank3-treated cultures. B-E, changes in the mean firing rate (B), bursting rate (C), intra-burst firing rate (D), and percentage of out-burst spikes (E) for active electrodes after the application of DHPG to shCtrl- and shShank3-treated cultures. There were no differences between shCtrl- and shShank3-treated groups in baseline activity; therefore, the values of parameters estimated under the baseline condition were set to 100% for each culture. Two-way ANOVA with repeated measures (DHPG concentrations of 1, 10, and 100 μm) revealed a significant effect of DHPG (p < 0.01), but not of shShank3 or shShank3 + DHPG on the mean firing rate (B). Significant effects of DHPG (p < 0.01) and shShank3 (p = 0.05) on the mean bursting rate were detected in C, and significant effects of shShank3 + DHPG (p < 0.05) on the intra-burst firing rate (D) and % of out-burst spikes (E) were detected. *, p < 0.05; **, p < 0.01, Holm-Sidak post hoc t test, significant differences between shCtrl- (n = 10 cultures) and shShank3-treated cultures (n = 7 cultures).CC BY-NC · PubMed Central
FIGURE 8. Restoration of frequency of mEPSCs and DHPG-induced phosphorylation of ERK1/2 in shShank3-treated cultured hippocampal neurons by the allosteric mGluR5 agonist CDPPB. A and B, neurons were infected with lentiviruses at 8 DIV and treated overnight with 1 μm CDPPB at 13 DIV. On 14 DIV, mEPSCs were recorded at a holding potential of −60 mV in the presence of 0.5 μm TTX and 50 μm picrotoxin. Summary graphs of the amplitude (A) and frequency (B) of mEPSCs in shCtrl + vehicle (n = 11 cells), shShank3 + vehicle (n = 14 cells), and shShank3 + CDPPB (n = 14 cells)-treated groups. Data are from four culture preparations. The frequency of mEPSCs is significantly decreased in shShank3 + vehicle as compared with shCtrl + vehicle-treated neurons and recovers significantly in shShank3 + CDPPB-treated cells; *, p ≤ 0.05, Dunn's test. Data are presented as mean ± S.E. C, Western blot of hippocampal neurons infected at 7 DIV with shCtrl or shShank3 and treated after 1 week as indicated above the panels. The proteins analyzed are indicated to the left of the panels. D, histogram showing mean ± S.E. for five independent experiments, of ERK1/2 phosphorylation, normalized to total ERK and untreated (NT) values. The level of pERK1/2 after DHPG treatment was significantly lower in the shShank3-infected neurons than in the shCtrl-infected neurons; *, p < 0.01, Student's t test.
FIGURE 8. Restoration of frequency of mEPSCs and DHPG-induced phosphorylation of ERK1/2 in shShank3-treated cultured hippocampal neurons by the allosteric mGluR5 agonist CDPPB. A and B, neurons were infected with lentiviruses at 8 DIV and treated overnight with 1 μm CDPPB at 13 DIV. On 14 DIV, mEPSCs were recorded at a holding potential of −60 mV in the presence of 0.5 μm TTX and 50 μm picrotoxin. Summary graphs of the amplitude (A) and frequency (B) of mEPSCs in shCtrl + vehicle (n = 11 cells), shShank3 + vehicle (n = 14 cells), and shShank3 + CDPPB (n = 14 cells)-treated groups. Data are from four culture preparations. The frequency of mEPSCs is significantly decreased in shShank3 + vehicle as compared with shCtrl + vehicle-treated neurons and recovers significantly in shShank3 + CDPPB-treated cells; *, p ≤ 0.05, Dunn's test. Data are presented as mean ± S.E. C, Western blot of hippocampal neurons infected at 7 DIV with shCtrl or shShank3 and treated after 1 week as indicated above the panels. The proteins analyzed are indicated to the left of the panels. D, histogram showing mean ± S.E. for five independent experiments, of ERK1/2 phosphorylation, normalized to total ERK and untreated (NT) values. The level of pERK1/2 after DHPG treatment was significantly lower in the shShank3-infected neurons than in the shCtrl-infected neurons; *, p < 0.01, Student's t test.CC BY-NC · PubMed Central

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Authors
Verpelli, C., Dvoretskova, E., Vicidomini, C., Rossi, F., Chiappalone, M., Schoen, M., Di Stefano, B., Mantegazza, R., Broccoli, V., Böckers, T. M., Dityatev, A., & Sala, C.
Affiliations
  • University of Milan (IT)
  • Italian Institute of Technology (IT)
  • Universität Ulm (DE)
  • Fondazione IRCCS Istituto Neurologico Carlo Besta (IT)
  • IRCCS Ospedale San Raffaele (IT)
Corresponding author
Sala, Carlo
ORCID
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2011-10
Volume
286
Issue
40
Pages
34839-34850
ISSN
0021-9258 (ISSN-L)
DOI
10.1074/jbc.m111.258384
PMC
PMC3186429
OpenAlex
W2073501423
Cited by
197
References
70
Citations by year
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FWCI
7.28
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • CINECA IRIS Institutial Research Information System (University of Genoa) (submittedVersion)
Funders
  • Fondazione Cariplo
  • Istituto Italiano di Tecnologia
  • Regione Lombardia
Topics
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetics and Neurodevelopmental Disorders
  • Life Sciences › Neuroscience › Cognitive Neuroscience › Autism Spectrum Disorder Research
Keywords
SHANK3, metabotropic glutamate receptor 5, postsynaptic density, synaptogenesis, dendritic spines, synaptic plasticity, long-term depression, ERK1/2 phosphorylation, CREB phosphorylation, DHPG, miniature excitatory postsynaptic currents, glutamatergic neurotransmission, positive allosteric modulators, autism spectrum disorder, intellectual disability
MeSH terms
Animals, Hippocampus, Mice, Inbred C57BL, Models, Biological, Nerve Tissue Proteins, Neurons, Phosphorylation, Synapses, Signal Transduction, Receptors, N-Methyl-D-Aspartate, Receptors, AMPA, Receptors, Metabotropic Glutamate, Adaptor Proteins, Signal Transducing, Dendritic Spines, Mice, Rats, Receptor, Metabotropic Glutamate 5

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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